Mutational analysis of cat-86 gene expression controlled by lactococcal promoters in Lactococcus lactis subsp. lactis and Escherichia coli.

Promoters were cloned from the chromosomal DNA of Lactococcus lactis subsp. lactis NP4510 by using promoter-probe vector pGKV210. N-Methyl-N'-nitro-N-nitrosoguanidine-induced mutagenesis of L. lactis-(pBV413), with low-level expression of the cat-86 gene, resulted in enhanced expression. Subcloning and sequencing of the mutated plasmid designated pBV415 revealed that the mutation is located within the PstI-HindIII fragment containing the coding sequence of the cat-86 gene (the 10th CTG codon was replaced by a TTG; both code for leucine). A set of otherwise identical plasmids with four combinations of CTG and TTG codons at the 10th and 46th positions in the cat-86 gene were constructed by site-directed mutagenesis. These plasmids containing cat-86 derivatives displayed a significant variation in cat expression in L. lactis and E. coli. The data suggest that cat expression is dependent on the secondary structure of the cat mRNA. New cat-86 derivatives described here can be used in lactococci, in which they provide additional flexibility for promoter cloning.     

Structure and expression of the Lactococcus lactis gene for phospho-beta-galactosidase (lacG) in Escherichia coli and L. lactis

The Lactococcus lactis subsp. lactis 712 lacG gene encoding phospho-beta-galactosidase was isolated from the lactose mini-plasmid pMG820 and cloned and expressed in Escherichia coli and L. lactis. The low phospho-beta-galactosidase activity in L. lactis transformed with high-copy-number plasmids containing the lacG gene contrasted with the high activity found in L. lactis containing the original, low-copy-number lactose plasmid pMG820, and indicated that the original lactose promoter was absent from the cloned DNA. In E. coli the phospho-beta-galactosidase could be overproduced using the strong inducible lambda PL promoter, which allowed a rapid purification of the active enzyme. The complete nucleotide sequence of the L. lactis lacG gene and its surrounding regions was determined. The deduced amino acid sequence was confirmed by comparison with the amino acid composition of the purified phospho-beta-galactosidase and its amino-terminal sequence. This also allowed the exact positioning of the lacG gene and identification of its characteristic Gram-positive translation initiation signals. The homologous expression data and the sequence organization of the L. lactis lacG gene indicate that the gene is organized into a large lactose operon which contains an intergenic promoter located in an inverted repeat immediately preceding the lacG gene. The organization and sequence of the L. lactis lacG gene were compared with those of the highly homologous lacG gene from Staphylococcus aureus. A remarkable bias for leucine codons was observed in the lacG genes of these two species. Heterogramic homology was observed between the deduced amino acid sequence of the L. lactis phospho-beta-galactosidase, that of the functionally analogous E. coli phospho-beta-glucosidase, and that of an Agrobacterium beta-glucosidase (cellobiase).     

腺苷酸脱氨酶在乳酸克鲁维酵母中构建表达

【目的】实现鼠灰链霉菌来源经密码子优化后的腺苷酸脱氨酶基因在乳酸克鲁维酵母(Kluyveromyces lactis GG799)中组成型表达。【方法】以鼠灰链霉菌(Streptomyces murinus)来源的腺苷酸脱氨酶(AMP)基因经密码子优化后作为模板,设计特异性引物,PCR扩增AMP脱氨酶基因opt-AMPD,以p KLAC1为载体构建重组表达质粒p KLAC1-opt-AMPD,经Sac II线性化后电转化法转入K.lactis GG799,筛选得到重组菌株,测定酶活,经His TrapTM HP纯化后得到AMP脱氨酶,并优化重组菌的发酵培养基。【结果】对AMP脱氨酶基因进行了密码子优化后,构建了重组K.lactis GG799/p KLAC1-opt-AMPD,实现组成型表达,密码子优化后AMP脱氨酶酶活提高到586±50 U/m L。SDS-PAGE结果显示,纯化后的AMP脱氨酶为单一条带,蛋白大小约为60 k D。优化的发酵培养基为(g/L):葡萄糖40、蛋白胨20、酵母粉15、Na Cl 8、KCl 10、Mg SO4 2,30°C、200 r/min发酵120 h,酶活达到2 100±60 U/m L。【结论】实现了密码子优化后的腺苷酸脱氨酶基因在乳酸克鲁维酵母GG799内的组成型表达,为实现腺苷酸脱氨酶的重组高效表达和发酵生产进行了有益探索。     

Codon distribution in vertebrate genes may be used to predict gene length

I have analysed the coding regions of 96 eukaryotic genes for their use of iso-coding codons. Specific codons occur more frequently in specific positions in all members of some gene families than would be expected if codon choice was determined solely by the frequency of codon usage. In the absence of evidence a priori for selection for particular codons at particular positions, I term such co-occurring codons “coincident codons”. Coincident codons are not confined to particular regions of genes, and their occurrence is not detectably linked with the location of introns in the genomic sequence. Their presence is partly but not completely explained by the exchange of sequence between similar functional genes within a species: homologous genes from different organisms also possess the same codons at some sites with greater than expected frequencies. The relative excess of coincident codons correlates well with the overall length of the genes analysed, but not with the length of mRNA or coding regions, or with qualitative features of gene structure or expression. This, and the unusual sequence environment of coincident codons, suggests that they are a feature of the overall secondary structure of the heterogeneous nuclear RNA. Such considerations suggest approaches for optimizing the expression of exogenous genes in eukaryotic systems, and for predicting the structure of genes for which only partial sequence data is available.     

Statistical evidence for conserved, local secondary structure in the coding regions of eukaryotic mRNAs and pre-mRNAs

Owing to the degeneracy of the genetic code, protein-coding regions of mRNA sequences can harbour more than only amino acid information. We search the mRNA sequences of 11 human protein-coding genes for evolutionarily conserved secondary structure elements using RNA-Decoder, a comparative secondary structure prediction program that is capable of explicitly taking the known protein-coding context of the mRNA sequences into account. We detect well-defined, conserved RNA secondary structure elements in the coding regions of the mRNA sequences and show that base-paired codons strongly correlate with sparse codons. We also investigate the role of repetitive elements in the formation of secondary structure and explain the use of alternate start codons in the caveolin-1 gene by a conserved secondary structure element overlapping the nominal start codon. We discuss the functional roles of our novel findings in regulating the gene expression on mRNA level. We also investigate the role of secondary structure on the correct splicing of the human CFTR gene. We study the wild-type version of the pre-mRNA as well as 29 variants with synonymous mutations in exon 12. By comparing our predicted secondary structures to the experimentally determined splicing efficiencies, we find with weak statistical significance that pre-mRNAs with high-splicing efficiencies have different predicted secondary structures than pre-mRNAs with low-splicing efficiencies.     

Translation of an atypical human cDNA requires fidelity of apurine-pyrimidine repeat region and recoding

Gain or loss of Migration inducting gene-7 (Mig-7) protein expression functional studies suggest it causes aggressive tumor cell invasion and tumor cell vessel-like structure formation. In addition, Mig-7 expression is apparently carcinoma and trophoblast cell-specific. Mig-7 is an example of an atypical gene that is unique in its induction, translation and apparent carcinoma-specific expression. However, studies of this predominantly integral membrane protein are hampered because of the cloning and expression techniques required for detection of Mig-7 protein. Because the encoding region possesses stop codons, repeat sequences and secondary structure, we hypothesized that genetically engineered E. coli are required to maintain the number of purine-pyrimidine repeats and reading frame when producing expression plasmids containing the Mig-7 sequence. Cloning Mig-7 sequence using E. coli genetically engineered to lack recombination and rearrangement capabilities prevented extension of the repeat region. Because of multiple stop codons in the sequence, three different constructs starting from three different reading frame ATG sites were tested for protein production in a human carcinoma cell line. Mig-7 protein of ~23 kD is produced from Mig-7 cDNA that contains multiple stop codons downstream from the ATG in a Kozak consensus sequence. In silico analyses imply that multiple Mig-7 mRNA secondary structures may cause frameshifting, read-through, and/or recoding of the multiple stop codons. Experimental results support that one or more of these translational events take place. In this report, we detail requirements for cloning and expression of this novel, atypical, human gene. These techniques can be used to express this unique protein for further studies.     

Codon bias at the 3'-side of the initiation codon is correlated with translation initiation efficiency in Escherichia coli

The codon that follows the AUG initiation triplet (+2 codon) affects gene expression in Escherichia coli. We have extended this analysis using two model genes lacking any apparent Shine-Dalgarno sequence. Depending on the identity of the +2 codon a difference in gene expression up to 20-fold could be obtained. The effects did not correlate with the levels of intracellular pools of cognate tRNA for the +2 codon, with putative secondary mRNA structures, or with mRNA stability. However, most +2 iso-codons that were decoded by the same species of tRNA gave pairwise similar effects, suggesting that the effect on gene expression was associated with the decoding tRNA. High adenine content of the +2 codon was associated with high gene expression. Of the fourteen +2 codons that mediated the highest efficiency, all except two had an adenine as the first base of the codon. Analysis of the 3540 E. coli genes from the TransTerm database revealed that codons associated with high gene expression in the two expression systems are over-represented at the +2 position in natural genes. Codons that are associated with low gene expression are under-represented. The data suggest that evolution has favored codons at the +2 position that give high translation initiation.     

The influence of mRNA primary and secondary structure on human IFN-gamma gene expression in E. coli.

Parameters influencing the efficiency of expression of the human immune interferon (IFN-gamma) gene in E. coli were studied by comparing a series of eight in vitro-derived gene variants. These contained all possible combinations of silent mutations in the first three codons of the mature IFN-gamma polypeptide coding sequence. Expression levels varied up to 50-fold among the different constructions. Comparison of messenger RNA secondary structure models for each variant suggested that the presence of stem-loop structures blocking the translation initiation signals could drastically decrease the efficiency of IFN-gamma synthesis. With variants displaying no stable mRNA secondary structure in the region, a C----U transition at position +11 after the AUG resulted in a 5-fold increase in expression indicating that RNA primary structure also plays an important role in expression. In addition we demonstrate that, in this system, a spacing of 8 nucleotides between the Shine-Dalgarno region and AUG was optimal for gene expression and that the steady-state production level of IFN-gamma rose exponentially with increasing rate of synthesis.     

Local absence of secondary structure permits translation of mRNAs that lack ribosome-binding sites

The initiation of translation is a fundamental and highly regulated process in gene expression. Translation initiation in prokaryotic systems usually requires interaction between the ribosome and an mRNA sequence upstream of the initiation codon, the so-called ribosome-binding site (Shine-Dalgarno sequence). However, a large number of genes do not possess Shine-Dalgarno sequences, and it is unknown how start codon recognition occurs in these mRNAs. We have performed genome-wide searches in various groups of prokaryotes in order to identify sequence elements and/or RNA secondary structural motifs that could mediate translation initiation in mRNAs lacking Shine-Dalgarno sequences. We find that mRNAs without a Shine-Dalgarno sequence are generally less structured in their translation initiation region and show a minimum of mRNA folding at the start codon. Using reporter gene constructs in bacteria, we also provide experimental support for local RNA unfoldedness determining start codon recognition in Shine-Dalgarno--independent translation. Consistent with this, we show that AUG start codons reside in single-stranded regions, whereas internal AUG codons are usually in structured regions of the mRNA. Taken together, our bioinformatics analyses and experimental data suggest that local absence of RNA secondary structure is necessary and sufficient to initiate Shine-Dalgarno--independent translation. Thus, our results provide a plausible mechanism for how the correct translation initiation site is recognized in the absence of a ribosome-binding site.     

Importance of mRNA folding and start codon accessibility in the expression of genes in a ribosomal protein operon of Escherichia coli.

The trmD operon of Escherichia coli consists of the genes for the ribosomal protein (r-protein) S16, a 21 kilodalton protein (21K) of unknown function, the tRNA(m1G37)methyltransferase (TrmD), and r-protein L19, in that order. The synthesis of the 21K and TrmD proteins is 12 and 40-fold lower, respectively, than that of the two r-proteins, although the corresponding parts of the mRNA are equally abundant. This translational control of expression of at least the 21K protein gene (21K), is mediated by a negative control element located between codons 18 and 50 of 21K. Here, we present evidence for a model in which mRNA sequences up to around 100 nucleotides downstream from the start codon of 21K fold back and base-pair to the 21K translation initiation region, thereby decreasing the translation initiation frequency. Mutations in the internal negative control element of 21K that would prevent the formation of the proposed mRNA secondary structure over both the Shine-Dalgarno (SD) sequence and the start codon increased expression up to about 20-fold, whereas mutations that would disrupt the base-pairing with the SD-sequence had only relatively small effects on expression. In addition, the expression increased 12-fold when the stop codon of the preceding gene, rpsP, was moved next to the SD-sequence of 21K allowing the ribosomes to unfold the postulated mRNA secondary structure. The expression increased up to 150-fold when that stop codon change was combined with the internal negative control element base-substitutions that derepressed translation about 20-fold. The negative control element of 21K does not seem to be responsible for the low expression of the trmD gene located downstream. However, a similar negative control element native to trmD can explain at least partly the low expression of trmD. Possibly, the two mRNA secondary structures function to decouple translation of 21K and trmD from that of the respective upstream cistron in order to achieve their independent regulation.     

Restructuring the translation initiation region of the human parathyroid hormone gene for improved expression in Escherichia coli

Overexpression of native human parathyroid hormone in Escherichia coli was achieved by a modification of the 5' end of the genomic gene sequence, thereby adapting this part of the translation initiation region to the bacterial host. Some simple rules abstracted from optimization studies of translation initiation of a beta-interferon gene were applied. These included (a) extending complementarity of the mRNA to the anticodon loop of tRNAfMet by use of a codon with a purine nucleotide directly following the ATG, (b) avoidance of stable secondary structure in the mRNA by use of synonymous A/U-rich codons, (c) elimination of a potential second Shine-Dalgarno sequence. The appropriate silent changes led to a 20-fold increase in parathyroid hormone production resulting in 4.3% of total soluble protein. This result proves the validity of our simple approach for optimization of foreign gene expression in E. coli.     

Antisense mRNA-Mediated Bacteriophage Resistance in Lactococcus lactis subsp. lactis

Resistance to a broad class of isometric bacteriophages that infect strains of Lactococcus lactis has been engineered into a dairy starter by expression of antisense mRNA targeted against a conserved bacteriophage gene. Maximum protection is obtained only when the entire 1,654-bp coding sequence for a 51-kDa protein is positioned in the antisense orientation with respect to a promoter sequence that functions in L. lactis subsp. lactis. Expression of the antisense mRNA results in more than 99% reduction of the total number of PFU. Plaques that do form are characterized by their relatively small size and irregular shape. A variety of truncated genes, including the open reading frame expressed in the sense orientation, fail to provide any significant measure of resistance as compared with that of the intact open reading frame. Southern hybridization with probes specific for the conserved region reveal that the [ill] plasmid constructs are maintained despite the presence of a large complement of other indigenous plasmids. Strains harboring the antisense mRNA plasmid construct grow and produce acid at a rate equivalent to that of the host strain alone, suggesting that antisense expression is not deleterious to normal cellular metabolism.